Animal reproduction requires the development of a functional germline, the set of highly specialized cells capable of passing on genetic material to the next generation. Throughout the animal kingdom, germline function and maintenance requires formation of large, complex ribonucleoprotein particles (RNPs) called germ granules. Germ granules control the post-transcriptional regulation of many mRNAs that are essential for germline differentiation, proliferation and maintenance. Evidence for the conserved role of germ granules comes from the effect of mutations that eliminate conserved germ granule components in Drosophila, Xenopus, zebrafish, and mouse, resulting in loss of primordial germ cells. Despite their importance, the underlying mechanisms that regulate germ granule formation are unclear; elucidating these mechanisms may provide insight into germline defects such as infertility and sterility. The proposed research investigates mechanisms regulating germ granule assembly using Drosophila germ granules, called polar granules, as a model. Results from a recent study show that polar granules comprise many different mRNAs. These mRNAs are proposed to co-assemble in a process in which mRNAs of a given gene interact to form multi-copy homotypic RNPs, then different homotypic RNPs of different transcripts associate to form higher-order polar granules that are heterogeneous in both the amount and types of mRNAs. Current data support two models of polar granule assembly: 1) polar granules form by the fusion of different homotypic RNPs or 2) homotypic RNPs of different mRNAs form concurrently on predefined scaffolding. The goal of Aim 1 is to test these models by using a combination of single molecule fluorescence in situ hybridization (smFISH), quantitative image analysis, and live imaging experiments. The molecular mechanisms regulating polar granule assembly are unknown and the analysis proposed in Aim 2 seeks to identify cis-acting RNA elements that mediate RNA granule assembly. Aim 3 focuses on the isolation and characterized of proteins that regulate the polar granule assembly process using RNA and protein affinity purification strategies combined with molecular and genetic techniques. Completion of the proposed aims will provide new insight into germ granule formation and its importance in germline development. Since different classes of RNA granules have shared features and conserved components, key findings from this work may have broader impact by application to other types RNA granules.